The overall goal of this proposal is to conduct late-stage preclinical studies to support a pilot clinical trial of hematopoietic stem cell transplantaton gene therapy for hemophilia A. In a series of recent studies, we have shown that the transplantation of genetically-engineered hematopoietic stem cells can restore factor VIII (fVIII) activity to curative levels in hemophilia A mice and that human hematopoietic stem cells are readily transduced with recombinant lentivirus encoding a genetically-engineered fVIII transgene. To date, we are the only group that has obtained sustained therapeutic fVIII expression levels in hemophilia A mice using non- myeloablative transplantation regimens that are in routine clinical practice. Our gene therapy approach uses a bioengineered fVIII transgene (termed ET-3) that achieves normal fVIII activity levels (1 unit/ml) at hematopoietic stem cell transduction efficiencies (1-5%), which are achieved currently in human gene therapy clinical trials. We have generated extensive preclinical data using ET-3 demonstrating proof-of-concept that hematopoietic stem cells genetically engineered with a lentivirus vector encoding ET-3, coupled with a non-myeloablative transplant regimen, can be used to treat hemophilia A. We now propose to first conduct pre-IND meetings with the FDA to direct final preclinical testing of ET-3. Therefore, our late stage testing will be based on FDA guidance. Second, we will generate our final preclinical data set using clinical- grade (GMP) lentiviral vector encoding ET-3, which will specifically test the safety and effectiveness of the clinical product. It is anticipted that a follow up phase II project will be submitted in support of the actual clinical trial. Four organizations have partnered to accomplish these goals, including: i) Expression Therapeutics, LLC, a biotechnology company founded on the high expression fVIII technology, ii) Emory University, where the conception and proof of concept of high expression fVIII technology occurred, iii) Children's Healthcare of Atlanta, financial supporter of the Gene Therapy Program at Emory University and investor in Expression Therapeutics, LLC, and iv) Lentigen Corporation, a company dedicated to the successful clinical application of lentiviral vectors and holder of the largest lentiviral vector intellectual property portfolio. Lentigen will generate the clinical-grade recombinant lentivector that will be used in the proposed studies including the clinical trial. PUBLIC HEALTH RELEVANCE: Insufficient expression of the blood clotting factor VIII results in the bleeding disorder hemophilia A. Current treatment for this disease consists of difficult, lie-long, intravenous infusion of plasma-derived or recombinant factor VIII to restore circulating factor VIII activity levels and is currently offered to less than one-third of all hemophilia A patients due to high product cost and limited availability. Gene therapy offers a potential cure fo this debilitating and, in many parts of the world, lethal disease. We have shown that transplantation of bone marrow cells genetically-modified to express an engineered factor VIII protein is a feasible treatment for hemophilia A. In the current application, we propose to conduct late-stage pre-clinical testing to support approval of a first in man clinical gene therapy trial.